Mapping of wine yeast and fungal diversity in the Small Carpathian wine-producing region (Slovakia): evaluation of phenotypic, genotypic and culture-independent approaches

The aim of this study was to investigate the yeast and fungal microflora diversity in wine-related samples of the cultivars Veltlin zeleny (Gruner Veltliner) and Frankovka modra (Blaufrankisch) produced in the Small Carpathian region (Slovakia) using different eukaryotic identification approaches. For phenotypic identification we performed auxotrophy profiling using the API 20C AUX kit (bioMerieux, Marcy l'Etoile, France) and culture on selective lysine medium. Genotypic identification was done by real-time PCR assays specific for different wine yeasts and by sequencing of the internal transcribed spaced (ITS) region (ITS1-5.8S rRNA-ITS2 region). An effective culture-independent approach was also employed in the form of analysis of the ITS2 region using fluorescence-ITS PCR (f-ITS PCR), cloning and sequencing. The ITS sequencing results were concordant with the results of the selective culture and real-time PCR assay, but for several strains they did not agree with the results of auxotrophy profiling. In the culture-independent approach, sequences of clones included 100 % matches to Hanseniaspora uvarum, Candida zemplinina, Metschnikowia pulcherrima, Aureobasidium pullulans, Saccharomyces cerevisiae and Issatchenkia terricola, as well as two novel sequences with a 5-bp difference to Sporobolomyces marcillae. The data from this study, and particularly the data from the culture-independent investigation, contribute to the picture of the wine microbial community of two cultivars important in wine production in Slovakia and Central Europe.